The advances in treatment notwithstanding, heart disease remains the number one cause of death in the Nation, claiming the lives of nearly one million Americans each year, and running up medical costs estimated at $286.5 billion per year. Ischemia-reperfusion injury is the major cause of complications of cardiac surgery;morbidity and mortality, but current cardioprotective options are very limited and suboptimal in high-risk patients. This leaves a dire need for effective treatments. Approaches to augment the cardioprotective actions of the physiological nucleoside adenosine (Ado) continue to hold promise and remain to be exploited for novel cardioprotective therapies. This project will pursue the identification of high-affinity specific inhibitors of a recently characterized novel pH-dependent cardiac adenosine transporter, ENT4. ENT4 selectively transports adenosine relative to other physiological nucleosides, making it an intriguing transporter to study with respect to adenosine modulation in myocardial ischemia and reperfusion. Currently, there are no known potent or specific inhibitors of ENT4 Ado transport. Thus, the identification of potent specific inhibitors is of high priority, not only for cardiac studies but for studying adenosine regulation by ENT4 in other tissues as well. They will facilitate studies of its role in cardiac ischemia-reperfusion, and show whether or not ENT4 could be a cardioprotection drug target for combination regimens with other cardioprotective agents like ENT1 nucleoside transport inhibitors. In line with our pursuit of adenosine uptake inhibitors as cardioprotective agents for the treatment of ischemia- reperfusion injury, we have cloned and stably expressed the human ENT4 transporter (hENT4) in a nucleoside transporter deficient porcine PK15 (PK15NTD) cell line. We subsequently used this expression system to screen, and identify small molecules inhibitors of ENT4 Ado transport with IC50s values down to submicromolar level, and selectivity up to 78-fold relative to one or the other human plasma membrane equilibrative nucleoside transporters, hENT1 or hENT2. Thus we aim to: 1) optimize the potency and selectivity of these lead compounds through structure- activity relationship (SAR), pharmacophore mapping and 3D-QSAR studies, and to 2) explore the cardioprotective effects of ENT4 inhibitors in an isolated rat heart global ischemia model of myocardial infarction. The new ENT4 inhibitors will be compared with our recently discovered cardioprotective ENT1 inhibitors (Zhu et al., Am. J. Physiol. Heart Circ. Physiol. 2007, 292, H2921-2926). We will explore the possibility that ENT4 inhibitors can be additive or synergistic in combination with ENT1 inhibitors for cardioprotection. A multidisciplinary approach combining parallel combinatorial synthesis, chemoinformatics/3D-QSAR, biochemistry, physiology and cell biology methods will be applied to achieve the aims of the project. The results will provide insights on the role of ENT4 in cardiac ischemia, and its inhibitors in cardioprotection, and provide potential lead compounds for the development of new cardioprotective drugs and/or use as much needed novel research tools to probe ENT4 physiology. The adenosine potentiation approach to cardioprotection could not be more timely, in light of recent clinical results showing that statins, which increase interstitial Ado levels by activating ecto-5'-nucleotidase, can reduce heart attack and stroke risks in people with normal cholesterol levels (Ridker et al., N. Engl. J. Med. 2008;AHA, 2008);and the demonstration of synergism between low dose statin and Ado uptake inhibitor for cardioprotection, when used in combination (Ye et al., 2007). PUBLIC HEALTH RELEVANCE: In this proposal, we seek to study a new class of adenosine uptake inhibitors as cardioprotective agents. We will investigate structure-activity relationships (SARs) and test the cardioprotective properties of the compounds in an isolated rat heart ischemia model.